Quantitative detection of periodontopathogens by real-time PCR

• Published in: Journal of microbiological methods Vol. 59; no. 1; pp. 117 - 125
• Main Authors: Nonnenmacher, Claudia, Dalpke, Alexander, Mutters, Reinier, Heeg, Klaus
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.10.2004; Elsevier Science
• Subjects: Aggregatibacter actinomycetemcomitans - genetics; Bacterial plant pathogens; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Dental Plaque - microbiology; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Fundamental and applied biological sciences. Psychology; Humans; Microbiology; Miscellaneous; Periodontitis - diagnosis; Periodontitis - microbiology; Periodontopathogens; Phytopathology. Animal pests. Plant and forest protection; Polymerase Chain Reaction - methods; Porphyromonas gingivalis - genetics; Prevotella intermedia - genetics; Real-time PCR; RNA, Ribosomal, 16S - chemistry; RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; Statistics, Nonparametric; Subgingival plaque; Taq Polymerase - metabolism; Real-time PCR; Subgingival plaque; Periodontopathogens; Still disease; Eubacteria; Diseases of the osteoarticular system; Clostridiales; Bacteroidaceae; Prevotella intermedia; Actinobacillus actinomycetemcomitans; Development; 16S-RNA; Bacteria; Quantitative analysis; Porphyromonas gingivalis; Stomatology; Sample; Real time; Standards; Pasteurellaceae; Plasmid; Polymerase chain reaction; Periodontal disease; Chronic; Periodontitis; Peptostreptococcus micros; Peptostreptococcaceae; Detection
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2004.06.006

Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10 2 to 10 8 and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples.